[Plasma and salivary acetylcholinesterase activity in amyotrophic lateral sclerosis]. / Aktivnost' atsetilkholinesterazy krovi i slyuny pri bokovom amiotroficheskom skleroze.

OBJECTIVE: Assessment of plasma and salivary acetylcholinesterase (AChE) activity in patients with amyotrophic lateral sclerosis (ALS) and in an animal model of the disease. MATERIAL AND METHODS: We studied 41 participants, aged 31 to 71 years, including 17 patients with diagnosed ALS (ALS group, average age 62.3±2.2), 9 patients with ALS mimics (disease control, average age 58.1±2.9), and 15 healthy people (normal control, average age 57.7±2.3). Plasma and salivary AChE activity was measured by using the Ellman colorimetric method. ALS severity was assessed using the ALSFRS-R scale. The King's College staging system and the Milano-Torino Scale (MiToS) were used to determine the stage of the disease. Transgenic FUS-mice were used as ALS model. RESULTS: Plasma AChE activity in the ALS group did not significantly differ from the control groups. There was also no significant correlation between plasma AChE activity and disease parameters such as the stage, duration, rate of progression, and severity. In transgenic FUS-mice plasma AChE activity also did not differ from wild-type mice. However, it has been shown that patients with ALS have significantly higher saliva AChE activity compared to normal controls. However, patients with the bulbar form of ALS had significantly higher values of salivary AChE activity compared to healthy controls. CONCLUSION: In patients with the bulbar form of ALS, an increase in salivary AChE activity was noted, which can be used for diagnostic and prognostic purposes. There is no significant change in plasma AChE activity in ALS patients.

[Biomarkers of amyotrophic lateral sclerosis]. / Biomarkery bokovogo amiotroficheskogo skleroza.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that has no effective treatment. To date, ALS is considered as a multifactorial heterogeneous disease, in which the death of motor neurons is a final result of the different pathological pathways. Modern diagnostic criteria and classification of ALS do not take into account all heterogeneity of the disease. Despite the development of molecular neurobiology and neurophysiology, genetics, and technology, significant progress in understanding the pathogenesis of ALS, the disease is diagnosed primarily on the basis of clinical manifestations. In recent years, a number of clinical trials of promising drugs have failed to show positive results. Among the reasons for these failures are variability of ALS forms, patients enrollment already at a late stage of the disease, the lack of use of biomarkers for patients selection and drugs' pharmacodynamics assessment. The study of biomarkers and their implementation in clinical practice can help to solve these problems. Here we will discuss the fluid-based biomarkers for ALS.

Dysfunction of Neuromuscular Synapses in the Genetic Model of Alzheimer's Disease.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

6-Methyluracil derivatives as acetylcholinesterase inhibitors for treatment of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the major age-related progressive neurodegenerative disorder. The brain of AD patients suffers from loss of cholinergic neurons and decreased number of synapses [1]. AD is caused by an imbalance between Aß production and clearance, resulting in increased amount of Aß in various forms [2]. Reduction of Aß production and increasing clearance of Aß pathogenic forms are key targets in the development of potential therapeutic agents for AD treatment. Unfortunately, only nosotropic approaches for treatment of AD are currently effective in humans. These approaches mainly focus on the inhibition of brain acetyl-cholinesterase (AChE) to increase lifetime of cerebral acetylcholine [3]. It is important to emphasize that AChE itself promotes the formation of Aß fibrils in vitro and Aß plaques in the cerebral cortex of transgenic mouse models of AD [4]. This property of AChE results from interaction between Aß and the peripheral anionic site of the enzyme (PAS) [5]. Dual binding site inhibitors of both catalytic active site (CAS) and PAS can simultaneously improve cognition and slow down the rate of Aß-induced neural degeneration. Unfortunately, the assortment of AChE PAS ligands is still extremely limited. OBJECTIVE: To study putative advantages of AChE non-charged PAS inhibitors based on 6-methyluracil derivatives for the treatment of Alzheimer's disease. METHODS: In vitro studies. Concentration of drug producing 50% of AChE/BuChE activity inhibition (IC50) was measured using the method of Ellman et al. [6]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [7]. The ability of compound to block in vitro AChE-induced Aß1-40 aggregation was studied using a thioflavin T (ThT) fluorescent probe [8].In vivo biological assays. For in vivo blood-brain barrier permeation assay brains were removed 30 min after IP injection of LD50 dose of tested compound injection. The inhibitory potency was measured using the method of Ellman.Scopolamine and transgenic models of AD were used to evaluate the influence of compound 35 on spatial memory performance.Water solution of scopolamine was injected to mice (ip) 20 minutes before starting memory test during 14 days [9]. Mice were assigned to 7 groups, including 4 groups receiving injection (ip) of compound in different dosages, donepezil-treated mice (donepezil is conventionally used to treat Alzheimer's disease), positive and negative control groups. Double transgenic (APP/PS1) mice expressing a chimeric mouse/human amyloid precursor protein and a mutant of human presenilin-1 [10] were assigned to 4 groups, including transgenic animals injected (ip) with compound 35 or donepezil solution, positive (transgenes injected with water) and negative (wild-type mice) controls.To evaluate spatial memory performance, mice were trained on a reward alternation task using a conventional T-maze [11]. The criterion for a mouse having learned the rewarded alternation task was 3 consecutive days of at least 5 correct responses out of the 6 free trials.For ß-amyloid peptide load was evaluated quantitatively as a number and summary area of Thioflavine S fluorescent spots in cerebral cortex and hippocampal images using Image J program. Statistical analyses were performed using the Mann-Whitney test. RESULTS: We evaluated the acute toxicity of the most active compounds. The most potent AChE inhibitor compound 35 (IC50 (AChE) = 5 ± 0.5 nM) exhibited the lowest LD50 values (51 mg/kg) and inhibited brain AChE by more than 71 ± 1%. Compound 35 at 10 nM, exhibited a significant (35 ± 9%) inhibitory activity toward human AChE-induced Aß aggregation.Scopolamine injection induced significant decrease in correct choice percentage in T-maze, as well as decrease in percentage of mice reaching criterion for learning the task by day 14. This memory deficit was relieved to some extent either by compound 35 (5 mg/kg) or donepezil (reference compound) treatment (0.75 mg/kg). Interestingly, higher doses of compound 35 (10 and 15 mg/kg) produced less therapeutic effect on spatial memory deficit.Group of APP/PS1 mice showed 3 times lower percentage of reaching behavioral criterion and lower percentage of correct choice in T-maze alternation task comparing to WT mice, whereas compound 35 (5 mg/kg) or Donepezil treatment effectively improved these parameters in APP/PS1 mice.Compound 35 treatment (5 mg/kg) during 14 days significantly reduced percentage of summary area and number of ß-amyloid peptide (ßAP) deposits visualized in sections of cerebral cortex, dentate gyrus, and hippocampal CA3 area in APP/PS1 mice. The most prominent reduction of ßAP load by compound 35 treatment was found in CA3 area and cerebral cortex. Meanwhile, Donepezil treatment (1 mg/kg) during 14 days significantly reduced ßAP load in cerebral cortex but not in dentate gyrus and CA3 area. CONCLUSIONS: Experiments showed that the most potent AChE inhibitor compound 35 (6-methyluracil derivative) permeated the blood-brain barrier, improved working memory in the APP/PS1 transgenic mice and significantly reduced the number and area of Aß plaques in the brain. Thus, compound 35 is a promising candidate as a bi-functional inhibitor of AChE for treatment of AD.

Human cognitive and neuro-psychiatric bio-markers in the cardiac peri-operative patient.

Some of the complexities of surgical interventions include neurological and psychiatric disturbances. Prompt identification and early treatment of these complications are pivotal in achieving excellent clinical results. Recognizing major adverse events such as stroke, seizure or delirium is usually straight-forward, however the discovery of less frequent or more subtle post-operative changes such as cognitive dysfunction might be delayed due to lack of appropriate diagnostic tools. This review summarizes biological markers that can be utilized as surrogates in evaluating surgery-related neuro-psychiatric disorders.

[The influence of beta-amyloid peptide on the functions of excitable tissues: physiological and pathological aspects].

Beta-amyloid peptide (betaAP) is a product of proteolytic cleavage of wide-spread transmembrane amyloid precursor protein. Betaap is physiological oligopeptide, which is present in biological fluids and tissues of healthy human organism at picomolar concentrations. However, excessive production, polymerization and accumulation of betaAP in tissues (first in neural tissue) underlie the pathogenesis of a number of neurodegenerative diseases (Alzheimer's disease and others). Although the presence of insoluble deposits of fibrillar betaAP (senile plaques) is a characteristic histopathological sign of Alzheimer's disease, soluble betaAP oligomers (dimers, trimers, etc.) possess most neurotoxicity. Soluble betaAP induces mitochondrial dysfunction, increased production of reactive oxygen species in neurons, disorganization of cell plasma membranes, disturbances of ion transport across cell membranes, impairment of synaptic transmission and long-term synaptic plasticity, etc. Recently a lot of attention is paid to possible peripheral effects of betaAP, related to its toxic influence on excitable structures of neuromuscular apparatus and cardiovascular system. At current review we represented state-of-art views on the processes of production and aggregation of betaAP in organism, cellular and molecular mechanisms of betaAP influence on physiological functions of excitable cells, the role of betaAP in pathogenesis of neurodegenerative diseases (Alzheimer's disease and others), as well as the results of our own studies in this field.

Analysis of the efficiency of gene-cell therapy in transgenic mice with amyotrophic lateral sclerosis phenotype.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.

Evaluation of post-surgical cognitive function and protein fingerprints in the cerebro-spinal fluid utilizing surface-enhanced laser Desorption/Ionization time-of-flight mass-spectrometry (SELDI-TOF MS) after coronary artery bypass grafting: review of proteomic analytic tools and introducing a new syndrome.

Cognitive dysfunction following surgery is a common complication, which increases the incidence of other co-morbid conditions, hospital and health-care costs. The reported rate of the occurrence of post-operative cognitive decline varies with different studies, depending on population profile, type of surgery, definition of cognitive disorder and detection methods, design of study, etc. It remains unclear whether these psychiatric signs and symptoms are direct results of the effects of surgery or general anesthesia. Nonetheless they are more frequent after cardiac surgery and are likely to be multi-factorial, but the patho-mechanisms are not yet fully characterized. This communication provides a synopsis of proteomics tools and delineates novel SELDI-TOF results to evaluate biomarkers in this regard. Presented for the first time is a classification of the clinically relevant forms of post-operative cognitive decline with the advent of a novel subclass.

Morphofunctional analysis of experimental model of esophageal achalasia in rats.

We carried out a detailed analysis of rat model of esophageal achalasia previously developed by us. Manifest morphological and functional disorders were observed in experimental achalasia: hyperplasia of the squamous epithelium, reduced number of nerve fibers, excessive growth of fibrous connective tissue in the esophageal wall, high contractile activity of the lower esophageal sphincter, and reduced motility of the longitudinal muscle layer. Changes in rat esophagus observed in experimental achalasia largely correlate with those in esophageal achalasia in humans. Hence, our experimental model can be used for the development of new methods of disease treatment.

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals.

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.